1. Field of the Invention
The field of the present invention is medical bio-engineering, and more particularly, pertains to ophthalmic applications in penetrating keratoplasty and cellular functions of human corneal endothelial cells.
2. Description of the Prior Art
Animal corneal endothelium such as rabbit, bovine, and feline, have been grown in cell culture using conventional techniques. The cultured cells closely resemble the parent, native endothelium. In culture, the endothelium synthesizes and deposits, in a polar fashion, a well organized basement membrane that contains molecules which are characteristic of all basement membranes. Cultured bovine corneal endothelial cells grown in tissue culture can be successfully transplanted back in vivo, resuming their normal pumping function. Feline corneal buttons coated with bovine corneal endothelial cells have been successfully transplanted in the same donor, resulting in functional endothelium. Limited success in higher primates has been demonstrated.
There is no cited evidence of human corneal primary or sub-cultured endothelium being successfully seeded onto either human or animal donors. Establishment of primary human corneal endothelial cell cultures were facilitated by outgrowth from isolated Descemet's membranes. This method of Descemet's dissection is technically difficult, with the additional disadvantage of introducing other contaminating cell types into the cell culture. Human endothelial cell growth from explants is often slow, as cells may take up to eight weeks to reach confluency. Enzymatic dissociation of human corneal endothelial cells from Descemet's membrane has resulted in the inability of these cells to reattach and form tight cell to cell junctions. Cellular attachment proteins were cleaved in the dissociation process unless extremely low levels of trypsin/EDTA were used. Establishment of primary human corneal endothelial cell cultures from donors over 20 years of age was limited or impossible. There is no cited evidence of primary cultures of either animal or human corneal cells being established from organ cultured tissue. Utilization of long term storage of human corneal endothelial cells at -80.degree. C. has not been demonstrated.
The human cornea is a complex organ composed of three different tissues separated from each other by acellular layers which have the biochemical characteristics of basement membranes. These layers can be defined as the corneal epithelium, the acellular Bowman's membrane, the corneal stroma, the acellular Descemet's membrane, and the most important, the corneal endothelium. This endothelial monolayer maintains the clarity of the cornea by actively pumping salts and water out of the connective tissue stroma and into the anterior chamber of the eye. The corneal endothelium is a monolayer composed of highly contact-inhibited, flattened cells with a hexagonal configuration. The apical surface of the corneal endothelium is exposed to the aqueous humor, a nutritive body fluid that constantly bathes these cells.
The human corneal endothelium has a limited regenerative capacity, and the success of penetrating keratoplasty in humans depends on transplanting an adequate amount of functioning donor corneal endothelium and its maintenance and survival post-keratoplasty.
The present invention reconstructs from isolated corneal endothelial cells, maintained in vitro, the corresponding enhanced, functional corneal endothelial monolayer on human donor corneas. This procedure requires that corneal endothelium must be specifically isolated from the other layers of the donor cornea. These isolated endothelial cells must be maintained in vitro, proliferated actively; and upon reaching confluence, the cell must maintain both the morphological and functional capabilities of these cells in vitro.